72 nicotinic acetylcholine receptors assemble, function, and are activated primarily via their 7-7 interfaces.
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We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of 7 and 2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged 7 and 2 subunits colocalize. Frster resonance energy transfer between fluorescently tagged subunits strongly suggested that 7 and 2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function 7 and 2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, 72 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of 7 nAChRs, although amplitudes of 72 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for 7 nAChRs. It is noteworthy that 72 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro--erythroidine that was not observed for 7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the 7-2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower 72 nAChR current amplitudes and challenges in identifying the function of native 72 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of 2 subunits within the 72 nAChRs.
Murray, T. A., Bertrand, D., Papke, R. L., George, A. A., Pantoja, R., Srinivasan, R., ... Lukas, R. J.
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Murray, Teresa A||Bertrand, Daniel||Papke, Roger L||George, Andrew A||Pantoja, Rigo||Srinivasan, Rahul||Liu, Qiang||Wu, Jie||Whiteaker, Paul||Lester, Henry A||Lukas, Ronald J
